We have been studying clathrin-independent forms of endocytosis (CIE) and have identified a number of endogenous PM proteins that enter cells through this mechanism. We have begun to study these proteins in detail in an attempt to understand how they travel in cells and whether they specifically interact with cellular machinery. To further understand the mechanism or mechanisms of CIE, we have embarked on an siRNA screen using the Dharmacon Trafficking Library that targets 140 human genes. We are looking for hits that affect endocytosis of two CIE cargo proteins: MHCI (major histocompatibility complex Class I) and CD59 (a GPI-lipid anchored protein). We are validating the hits and will follow up on promising ones. Interestingly, we find that the hits that affect MHCI vs. CD59 endocytosis are different, suggesting that the requirements for internalization of these 2 CIE cargo proteins are somewhat distinct. In another study, we were examining the role of glycosylation on cargo proteins and whether over or under glycosylation will affect rates of endocytosis and the trafficking of those proteins. We examined endocytosis of two CIE cargo proteins - MHC Class I and CD59 - and found that increased glycosylation stimulated MHCI endocytosis but inhibited CD59 endocytosis. Furthermore exogenous galectin-3, a cellular lectin that binds to glycoproteins on the surface, enhances MHCI but inhibits CD59 endocytosis (Mathew and Donaldson, 2018). Thus, changes in glycosylation of cargo proteins and their interactions with galectins affects internalization of CIE cargo proteins in unique and specific ways. This provides a new paradigm for how CIE cargo enter cells and may explain the huge variety of reported types of CIE and CIE cargo proteins.